The recovery of large proteinaceous material through a small pore exclusion filter during purification, viral reduction or viral clearance processing has posed significant problems for the pharmaceutical and diagnostic industry (see Roberts, P., Vox Sang, 1995;69:82-83). Removal of virus from a large biomolecule such as a gamma globulin (monoclonal or polyclonal) by size exclusion is hindered by the difficulty of efficiently passing a large globular protein through a size exclusion filter with a 12-15 nm pore size. The problem is especially evident where small non-enveloped virus are sought to be removed from products with high molecular weight. A further complication to the process is that proteinaceous materials such as immunoglobulins to be recovered form dimers and trimers, which pass through the filters with difficulty or not at all.
The smaller the membrane pore size the more effective the membrane is in retaining the viral particles. However, along with the smaller pore size comes a decrease in the ability of the membrane to allow the virally cleared product to freely pass through. As with all virus-reduction technologies each application must be assessed on its own merits for each product and each virus. In those cases where the removal of small non-enveloped viruses is required, the use of virus filters with the smallest pore size (less than 35 nm, and preferably between 12-30 nm) is probably essential and this may not be possible with products of high molecular weight, or with those that form dimers and trimers.
The problem is outlined in the publication of Roberts referenced hereinabove wherein it is discussed that while filters with nominal cutoff values of 70, 160 kD, and 15, 35, 40, 50 and 70 nm may be useful for removing small viruses and prions, products with larger molecular weights such as immunoglobulins (IgG, 150 kD) and factor VIII (350 kD) can only pass through filters of larger pore size.
The methods of the invention comprise the filtration processing and viral clearance of a pharmaceutical preparation of an immunoglobulin.
In the prevention of hemolytic disease of newborn the mother is injected with Rho(D) immunoglobulin of human origin. Such a product is RHOGAM.RTM. Rho(D) Immune Globulin (Human), available from the assignee hereof, and it operates by preventing the unimmunized Rho (D) negative mother from responding to Rho (D) antigen present on red cells and `received` from an Rho(D) positive infant. Thus, by preventing anti-Rho (D) production by the mother, the subsequent Rho (D) positive infant of this mother is protected from hemolytic disease of the newborn. Although this successful product is presently produced by a Cohn alcohol fractionation type process, several investigators have attempted to use alternative methods to produce similar materials to thereby provide an economically more advantageous product, to reduce large plasma requirements. Such investigational efforts have been reported by Hoppe et al. in "Prevention of Rh Immunization Modified Production of IgG Anti-Rh For Intravenous Application By Ion Exchanged Chromatography", Vox Sang, 25:308-316 (1973) and Friesen et al. in "Column Ion-Exchange Preparation and Characterization of an Rh Immune Globulin for Intravenous Use", Journal of Applied Biochemistry, 3, 164-175 (1981).
Hoppe in Germany and Friesen in Canada both employed a DEAE-Sephadex chromatography column in conjunction with a phosphate buffer eluding agent. Hoppe's source of anti-D containing plasma was from volunteers who passed an HB Ag laboratory test for at least six months, the plasma being stored in the interim. Thus, Hoppe employed a relatively safe, noninfective plasma to start with. No additional tests were run, however, to determine the efficacy of the DEAE-Sephadex hepatitis B surface antigen. Hoppe's concern was instead directed towards the removal of aggregated materials and the isolation of an unfragmented, immunoeletrophoretically pure IgG having a relatively high antibody concentration. The Friesen publication reports on the modification's made to the Hoppe method for the development of an intravenous Rh IgG for use in Canada. As Hoppe had done, Friesen tested each unit of Rh plasma for HB AG to eliminate any donors testing positive. Friesen employed the radioimmunoassay kit from Abbott Laboratories, North Chicago, Ill. (Ausria I Kit). This test is still regarded as one of the most sensitive and was also employed in the development of the invention described later. Friesen reported that clinical trials showed the material produced using the DEAE Sephadex resin/phosphate buffer combination was effective and safe for the prevention of Rh immunization. Friesen, however, reported no additional tests for determining the efficacy of the DEAE-Sephadex/phosphate buffer combination for removing hepatitis B surface antigen from plasma samples. This, at least from the U.S. government's perspective, is especially important since the radioimmunoassay test employed in screening the donor plasma samples is incapable of detecting concentrations of HB AG particles two or three orders of magnitude lower which may still be infective. It is this concern for the potential infectivity of a reagent produced by such a method that the United States government has been significantly more restrictive in permitting the production of injectable immunoglobulin in reagents by solid phase methodologies.
RhoGAM.RTM. Rho(D) Immune Globulin (Human) was the first successful prophylactic use of specific antibody to achieve antibody mediated immune suppression. RHOGAM.RTM. Rho(D) Immune Globulin (Human) is an IgG immunoglobulin solution containing anti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose. RHOGAM.RTM. Rho(D) Immune Globulin (Human) can be given to the nonimmunized, Rho(D) negative pregnant woman at the appropriate time prevent future disease in her Rho(D) positive offspring. The disease is called hemolytic disease of the newborn or more specifically, Rh-erythroblastosis fetalis.
A smaller dose of anti-Rho(D), MICRHOGAM.RTM. Rho(D) Immune Globulin (Human) (50 micrograms of anti-Rho(D)) is also sold by the Assignee hereof for treatment of women who have abortions and miscarriages at twelve weeks gestation or earlier. While the full dose protects the recipient for up to 15 ml of Rho(D) positive red cells, the smaller dose provides protection up to 2.5 ml of Rho(D) positive red cells. RHOGAM.RTM. Rho(D) Immune Globulin (Human) is used as antenatal prophylaxis at 26 to 28 weeks gestation. Other indications include threatened abortion at any stage of gestation with continuation of pregnancy, abortion or termination of pregnancy at or beyond 13 weeks gestation, abdominal trauma or genetic amniocentesis, chorionic villus sampling (CVS) and percutaneous umbilical blood sampling (PUBS).
Most immunoglobulin injectable materials approved for use by the FDA and Bureau of Biologics have been produced by the alcohol fractionation procedure developed by Dr. E. Cohn of Harvard during the 1940s and described in Cohn et al., J. Am. Chem. Soc. 68, 459 (1946), incorporated herein by reference. This procedure coupled with the careful selection of plasma negative for hepatitis infectivity, HIV, and other blood-borne pathogens determined by the most sensitive tests available, has been employed for such a long period of time that the US government has adopted a position favoring only the resultant preparation of this procedure as safe. That the products produced by this procedure are indeed safe can easily be demonstrated by the millions of non-infected recipients of product.
Several conventional methods for the separation of gamma globulin from human serum have been described notably for Baumstark et al. in "A Preparative Method For The Separation of 7S Gamma Globulin From Human Serum", Archives of Biochemistry and Biophysics, 108, 514-522 (1964) and by A. Webb in "A 30-Minute Preparative Method For Isolation Of IgG From Human Serum", Vox Sang, 23:279-290 (1972), both of which are incorporated herein by reference. Although both of these papers are more concerned with the separation and selection of various gamma globulin classes from a serum containing numerous other contaminating proteins, they do address the removal of contaminating proteins and materials from the original serum sample. Both employ a DEAE-Sephadex column chromatographic material with a phosphate buffer eluting agent. Both investigators met with some degree of success as far as removal of contaminating proteins was concerned, however, both failed to address the problem of removing contaminating hepatitis viral particles in order to provide a safe, injectable reagent.
It is an object of the instant invention to provide virally cleared, pure immunoglobulin for injection. Such a substantially pure product is produced using the processing methods of the invention.
It is a further object of this invention to provide a manufacturable process for purifying immunoglobulins which is reasonable in terms of temporal, square foot and protein yield requirements.
The filtration used in this invention is accomplished by a sieve-retention mechanism dependent upon the size relationship of the virus to the filter mean pore size. Its efficiency is not affected by filtration conditions of temperature, ionic strength, virus titer challenge, pressure, pH, surface tension, and other variables. While affecting the ability of the IgG particle to pass through the filter, the detergent and ionic strength conditions of the invention do not affect the viral clearance. Studies conducted for the Assignee hereof have shown that the buffer composition employed has minimal effect on the virus particle in terms of viral inactivation and viral envelope removal.